2.1. Dog morphotype samples

We attempted to study the Amy2B copy number, using ancient DNA (aDNA) analysis from the tooth and bone remains of 88 different canids from 30 archaeological sites in Western Europe, Romania, Russia, Estonia, Israel, Turkmenistan and the Iranian Plateau, from the Upper Palaeolithic to the Bronze Age. In total, aDNA results were obtained from 13 individuals from eight archaeological sites in Europe and Turkmenistan (see the electronic supplementary material). The osteological distinction between the domestic dog (Canis familiaris) and the wolf (Canis lupus), its wild ancestor, can be difficult, due to the regional and temporal variability of wolf morphology [22] and to the morphological proximity between the two forms in the early steps of domestication; therefore, we used a series of osteological traits to separate them [3,23,24]. Dogs differ from wolves by their overall significantly smaller size, a smaller brain-case volume, a shorter snout, tooth crowding and a higher frequency of dental defects. All the individuals used in this study belonged to the domestic form, according to one or several of these criteria.

When possible, measurements were taken from mandibles, particularly the five dimensions frequently measurable in broken archaeological specimens (dimensions #8, 10, 11, 19, 20, after [25]; electronic supplementary material, table S1—only measurements for individuals providing aDNA results are reported). The data obtained for our archaeological Holocene canids were then compared with the data derived from (i) a series of Pleistocene wolf mandibles from Arcy-sur-Cure (France) [23] dated between 100 000 and 60 000 years BP, prior to any suspicion of domestication; (ii) a series of Pleistocene canid mandibles from Předmostí (Czech Republic) [26], attributed to the wolf and dated to 27 000–26 000 BP; (iii) a series of modern Eurasian wolf mandibles from the National Museum of Natural History, Paris [23]; and (iv) a series of modern wolf mandibles from Southeastern Europe [27] (electronic supplementary material, figure S1). It was noted that the length of the tooth row (dimension #8 [25]) was significantly different between the Holocene canids and the four series of wolf (Mann–Whitney tests corrected for Bonferroni, p < 0.05). The only individual in the Holocene series, located at the very margin of the modern wolves' variation interval (CH1075; electronic supplementary material, figure S1), evidenced a colour mutation typical to domestic animals from one of our previous studies on the same material [15]. Therefore, the canid series analysed in this study can be identified to be the domestic form C. familiaris.